The generation of molecules with high affinity and specificity for biological targets is a central problem in chemistry, biology and pharmaceutical sciences. In particular, binding ligands are important for the creation of drugs that can intervene with biological processes. The creation of ligands that bind to a chosen target ligand usually involves a process of generating a plurality of putative binding molecules and testing said molecules for their binding properties.
While biological in vitro selection techniques were used efficiently for the isolation of large biopolymeric structures such as antibodies, they were less practicable for the isolation of small molecule drugs to date. Biological in vitro selection techniques are generally limited to biological polymers such as polypeptides, RNA or DNA. Short biopolymers as for example peptides can also bind to biological targets but they can suffer from conformational flexibility and may be prone to proteolytic degradation in bodily fluids. In addition, binding affinities of short linear peptides are often weak. Various circularization strategies are known to constrain genetically encoded small peptide libraries. Phage displayed peptide repertoires are known for example to be circularized by the oxidation of two flanking cysteine residues. mRNA encoded cyclic peptide libraries are known to be generated by linking the N-terminal amine and a lysine residue of the peptide with a chemical cross-linking reagent. This strategy was used for the isolation of redox-insensitive macrocycles that bind to the signaling protein Gαil (Millward, S. W. et al., ACS Chem. Biol., 2007). Various strategies are also known for use to incorporate non-natural building blocks into genetically encoded polypeptide libraries to expand the diversity of the libraries or to insert properties that can not be provided by natural amino acids. However, the strategies allowed only the addition of a limited number of small organic appendages to linear genetically encoded polypeptides. Frankel, A. et al., for example had incorporated non-natural amino acids into natural polypeptides that were encoded by mRNA display (Frankel, A. et al., Chem. Biol., 2003). Jespers L. et al. had chemically linked a fluorescent reporter molecule to a hypervariable loop of an antibody repertoire displayed on phage, and selected this repertoire for antigen binding (Jespers, L., et al., Prot. Eng., 2004). Dwyer, M. A. et al. had joined synthetic peptides to a repertoire of phage displayed peptides by native chemical ligation for the generation of a protease inhibitor library containing a non-natural amino acid (Dwyer, M. A. et al., Chemistry & Biology, 2000). Small organic molecules have also been linked to mRNA encoded combinatorial peptide repertoires. The research team of Roberts, R. W. had attached a penicillin moiety to a fixed position of an mRNA-display peptide library to select inhibitors of the Staphylococcus aureus penicillin binding protein 2a (Li, S. and Roberts, W. R., Chem. & Biol., 2003).
In order to apply in vitro selection to combinatorial compound libraries having more diverse molecule architectures (e.g. branched molecules) and being formed of non-natural building blocks, various methodologies have been proposed. Unlike biological in vitro selection methods, these methodologies use chemical strategies to attach DNA tags to small organic molecules. Brenner S. and Lerner R. A. had proposed a process of parallel combinatorial synthesis to encode individual members of a large library of chemicals with unique nucleotide sequences on beads (Brenner, S. and Lerner, R. A., PNAS, 1992). After the chemical entity is bound to the target, the genetic code is decoded by sequencing of the nucleotide tag. Liu D. R. and co-workers had conjugated a small collection of organic molecules to DNA oligonucleotides and performed affinity selections with different antigens (Doyon, J. B. et al., JACS, 2003). Neri D. and co-workers had generated large repertoires of molecule pairs by self-assembly of smaller DNA encoded chemical sub-libraries through hybridization of two DNA strands (Melkko, S. et al., Nature Biotechnol., 2004). The methodology was successfully used for affinity maturation of small molecule ligands. Halpin D. R. and Harris P. B. developed a strategy for the in vitro evolution of combinatorial chemical libraries that involves amplification of selected compounds to perform multiple selection rounds (Halpin, D. R. and Harbury, P. B., PLOS Biology, 2004). Woiwode T. F. et al. attached libraries of synthetic compounds to coat proteins of bacteriophage particles such that the identity of the chemical structure is specified in the genome of the phage (Woiwode, T. F., Chem. & Biol., 2003). All these strategies employing DNA specified chemical compounds have proven to be efficient in model experiments and some have even yielded novel small molecule binders. However, it became apparent that the encoding of large compound libraries and the amplification of selected compounds is much more demanding than the equivalent procedures in biological selection systems.
Jespers et al (2004 Protein engineering design and selection, volume 17, no. 10, pages 709-713) describes the selection of optical biosensors from chemisynthetic antibody libraries. This document is concerned with the attachment of a fluorescent reporter molecule through the hypervariable loop of an antibody repertoire displayed on the phage. In particular, this document describes linking of a fluorescent reporter molecule into a hypervariable loop (complementarity determining region or CDR) of a synthetic antibody repertoire. The fluorescent reporter molecule is linked by a single covalent bond to an artificially introduced cysteine residue in the hypervariable loop. A one to one attachment is performed. The cysteine residues on the phage particles were reduced with DTT and the excess reducing agent was removed by conventional polyethylene glycol (PEG) precipitation as is well known in the art.
Dwyer et al disclose biosynthetic phage display, describing a novel protein engineering tool combining chemical and genetic diversity. Dwyer et al (Chem Biol 2000, volume 7, no. 4, pages 263-274) describe the chemical ligation of a synthetic peptide having a non-natural amino acid onto a library of synthetic peptides comprising the main structural residues of a protein of interest. The motivation for performing this was in order to generate a diverse range of protease sequences, each having a constant segment incorporating an unnatural amino acid. The synthetic peptide comprising the non-natural amino acid was simply joined by native chemical ligation, resulting in coupling of the two peptide fragments together. No connector compound is disclosed. No small molecule attachment is disclosed. No constraint or conformational restriction of the resulting polypeptide was achieved. No covalent bonding of particular moieties to the polypeptide chain is disclosed.
Different research teams have previously tethered polypeptides with cysteine residues to a synthetic molecular structure (Kemp, D. S. and McNamara, P. E., J. Org. Chem, 1985; Timmerman, P. et al., ChemBioChem, 2005). Meloen and co-workers had used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclisation of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman, P. et al., ChemBioChem, 2005). Methods for the generation of candidate drug compounds wherein said compounds are generated by linking cysteine containing polypeptides to a molecular scaffold as for example tris(bromomethyl)benzene are disclosed in WO 2004/077062 and WO 2006/078161.
Methods provided in WO 2004/077062 and WO 2006/078161, are based on sampling individual compounds for example in a screening procedure. Screening of individual compounds or small sets of compounds is tedious and can be expensive if large numbers of compounds are analyzed. The number of compounds that can be assayed with screening assays generally does not exceed several thousands. Moreover, reaction conditions described in WO 2004/077062 to tether a cysteine containing peptide to a halomethyl containing scaffold as for example tris(bromomethyl)benzene are not suitable to modify a genetically encoded cysteine containing peptide.
WO2004/077062 discloses a method of selecting a candidate drug compound. In particular, this document discloses various scaffold molecules comprising first and second reactive groups, and contacting said scaffold with a further molecule to form at least two linkages between the scaffold and the further molecule in a coupling reaction. This method suffers from many restrictions. Firstly, it is based on the use of synthetic peptides and in vitro chemical reactions in separate vessels. For this reason, it is labour intensive. There is no opportunity to automate or to apply the method to the screening of many peptide variants without manually producing each variant by conducting numerous parallel independent reactions. There is no mention of genetically encoded diversity in this document, and certainly no mention of application to genetically encoded phage libraries. Indeed, the reaction conditions disclosed in this document mean that it would be difficult or impossible to perform the reactions disclosed on phage particles.
WO2006/078161 discloses binding compounds, immunogenic compounds and peptidomimetics. This document discloses the artificial synthesis of various collections of peptides taken from existing proteins. These peptides are then combined with a constant synthetic peptide having some amino acid changes introduced in order to produce combinatorial libraries. By introducing this diversity via the chemical linkage to separate peptides featuring various amino acid changes, an increased opportunity to find the desired binding activity is provided. FIG. 7 of this document shows a schematic representation of the synthesis of various loop peptide constructs. There is no disclosure of genetically encoded peptide libraries in this document. There is no disclosure of the use of phage display techniques in this document. This document discloses a process which is considered to be incompatible with phage display. For example, the chemistry set out in this document is likely to result in the linking molecule reacting with the phage coat. There is a risk that it could cross link phage particles. It is probable that phage particles would be inactivated (e.g. would lose their infectivity) if subjected to the chemistry described. This document is focussed on the manipulation of various synthetic peptides in independent chemical conjugation reactions.
Millward et al (2007 Chemical Biology, volume 2, no. 9, pages 625-634) disclose the design of cyclic peptides that bind protein surfaces with antibody like affinity. This document discloses cyclisation of various peptides produced from a genetically encoded library. The polypeptides are cyclised through reaction of a chemical cross-linker with the N-terminal amine and an amine of a lysine in the polypeptide. In this document, the genetically encoded library is a mRNA display library. This document does not disclose the attachment of any connector compound to the resulting polypeptides. This document is concerned with the production of redox insensitive cyclised peptides. The chemistry disclosed in this document is cyclisation through reaction of a chemical cross linker with the N-terminal amine and an amine of a lysine provided in the polypeptide. The cyclisation reaction is performed in a 50 milimolar phosphate buffer at pH 8 by the addition of DSG (1 mg per ml in DMF). At most, this document discloses the bridging of two parts of a polypeptide chain via a cross linking moiety in order to provide a cyclic peptide.
US2003/0235852 discloses nucleic acid-peptide display libraries containing peptides with unnatural amino acid residues, and methods of making these using peptide modifying agents. In other words, this document discloses genetically encoded polypeptide libraries that contain either a non-natural amino acid or an amino acid where a non-natural building block (e.g. penicillin) is post-translationally attached in a chemical reaction. This document is focused on known methods for associating a translated peptide with the nucleic acid which encoded it. The further problem addressed by this document is how to incorporate unnatural amino acids into that peptide. This is principally accomplished by the use of suppressor tRNAs in order to incorporate unnatural amino acids in response to amber/ochre/opal codons as is well known in the art. In other more minor embodiments, unnatural amino acids are created post-translationally by treatment of the translated peptide with a ‘peptide modifying agent’. This reagent is typically aimed at altering an existing amino acid residue in order to convert it into an unnatural amino acid residue, or otherwise render it functionally reactive or receptive to the attachment of a further chemical moiety. Specifically, this document teaches the post-translational conjugation of a cysteine residue in the polypeptide of interest to the beta lactam antibiotic 6-bromoacetyl penicilamic acid. This results in the conjugation of this penicillin analogue onto the polypeptide of interest via a single bond to the cysteine residue side chain. No multiple bonding of the molecule being ligated to the polypeptide is disclosed. No conformational constraint of the polypeptide is described. No peptide loops or any other complex tertiary structures are formed by the methods disclosed in this document—it is purely a way of attaching a single further molecular group to a polypeptide via a single bond. Conventional conjugation chemistry is used in order to perform the modifications to the polypeptides in this document.